Although many therapeutic strategies exist for molecular targets accessible from the outside of the cell (e.g. therapeutic antibodies) or within the cytoplasm (e.g. small molecule inhibitors), they are not applicable to molecular targets that lie within the membrane bilayer. The hydrophobic phospholipid bilayer imposes an impenetrable barrier to water-soluble polar therapeutic agents. The Yin lab recently developed a computational method, Computed Helical Anti-Membrane Protein (CHAMP), to rationally design peptide probes that recognize protein transmembrane domains (TMDs) with high affinity and selectivity. Nonetheless, all previous work has been limited to single-pass TMDs. Among the membrane-associating proteins that account for approximately 25% to 30% of the human proteome, multi-spanning membrane proteins are particularly challenging to study due to the high degree of difficulty involved in preparing, characterizing, and analyzing these transmembrane proteins. With the proposed studies, we aim to develop a generally applicable method to study previously inaccessible multi-spanning membrane protein associations. Latent Membrane Protein 1 (LMP-1) was chosen as a model system to test this technology because of the essential role of its multi-spanning TMD in activation of signaling and its clinical relevance, particularly to lymphoid malignancies and lymphoproliferative syndromes associated to human Epstein-Barr virus (EBV). EBV's ability to infect and immortalize B lymphocytes depends on the expression and activity of LMP-1, the multi-spanning, viral oncoprotein expressed in many EBV-dependent lymphomas and lymphoproliferative syndromes. LMP-1 most resembles the Tumor Necrosis Factor Receptor (TNFR) CD40 in its signaling. Unlike CD40, whose activity requires activation by ligand, LMP-1's activity is constitutive and ligand-independent. Constitutive homo-oligomerization of LMP-1's TMD plays a key role in activation of downstream signaling. This study aims to develop an innovative approach to target LMP-1's TMD, using CHAMP-designed anti-TMD peptide antagonists as probes to study the contribution of oligomerization to LMP-1 activation, with the goal of inhibiting downstream signaling. To the CHAMP method, we will introduce a novel screening dimension as well as a next generation of algorithm. Results of this research will provide insight into the molecular interactions within the membrane environment and the mechanisms underlying constitutive/oncogenic receptor signal transduction across membranes, will reveal the mechanism of LMP-1's constitutive activation of signaling, and will be applicable to the future development of novel therapeutics targeting diseases dependent on critical multi-spanning transmembrane proteins. Specifically, this proposal addresses the following Aims: 1) Develop specific peptide probes targeting individual TMDs of LMP-1; and 2) Determine the role of TMD-mediated oligomerization in LMP-1 activation.